A temperature-sensitive lethal mutant of Staphylococcus aureus was found to harbor a mutation in the uncharacterized two-component histidine kinase (HK)-response regulator (RR) pair encoded by yycFG; orthologues of yycFG could be identified in the genomes of Bacillus subtilis and other gram-positive bacteria. Sequence analysis of the mutant revealed a point mutation resulting in a nonconservative change (Glu to Lys) in the regulator domain of the RR at position 63. To confirm that this signal transduction system was essential, a disrupted copy of either the RR (yycF) or the HK (yycG) was constructed with a set of suicide vectors and used to generate tandem duplications in the chromosome. Resolution of the duplications, leaving an insertion in either the yycF or the yycG coding region, was achieved only in the presence of an additional wild-type copy of the two open reading frames. Phenotypic characterization of the conditional lethal mutant showed that at permissive growth conditions, the mutant was hypersusceptible to macrolide and lincosamide antibiotics, even in the presence of the ermB resistance determinant. Other mutant phenotypes, including hypersensitivity to unsaturated long-chain fatty acids and suppression of the conditional lethal phenotype by high sucrose and NaCl concentrations, suggest that the role of the two-component system includes the proper regulation of bacterial cell wall or membrane composition. The effects of this point mutation are strongly bactericidal at the nonpermissive temperature, indicating that this pathway provides an excellent target for the identification of novel antibiotics.
We isolated mutations that reduce plasmid stability in dividing cell populations and mapped these mutations to a previously undescribed gene, recD, that affects recombination frequency and consequently the formation of plasmid concatemers. Insertions of the transposable element TnlO into recD resulted in increased concatemerization and loss of pSC101 and ColEl-like replicons during nonselective growth. Both concatemer formation and plasmid instability in recD mutants require a functional recA gene. Mutations in recD are recessive to recD+ and map to a small region of the Escherichia coi chromosome located between recB and argA. Although the recD locus is distinct from loci encoding the two previously identified subunits of the RecBC enzyme, mutations in recD appear to affect the exonuclease activity of this enzyme.The maintenance of plasmids as extrachromosomal elements in bacterial cells is dependent on a number of complex processes. Of primary importance is the requirement for plasmid replication, which commonly involves at least some host-encoded replication functions (see Scott [39] for a review). Replication of the pSC101 plasmid (7) requires the Escherichia coli dnaA gene product (21). Mutations in other genes involved in chromosomal replication, including dnaB, dnaC, and dnaG (14,22), also affect replication of this plasmid. Stable maintenance of pSC101 in growing cell populations also requires a plasmid locus (named par for partitioning) that is involved in the distribution of plasmid molecules to daughter cells at the time of cell division (29,45). Earlier work has shown that the pSC101 par region does not encode a protein but instead includes partition-related segments (30) that appear to be necessary for plasmids in the intracellular pool to be counted and partitioned as individual molecules (45). To better understand the mechanism by which pSC101 segregates during cell division, we undertook to identify and characterize E. coli genes encoding products involved in the plasmid maintenance (Pma) phenotype.We report here the isolation and characterization of mutations that affect the stable maintenance of pSC101, as well as that of certain other plasmids. We present evidence that these mutations, which can alter plasmid stability by increasing the frequency of multimer formation and are located in a previously unidentified E. coli gene, affect the activity of exonuclease V (ExoV). MATERIALS AND METHODSStrains, media, and general methodology. Relevant bacterial genotypes, plasmids, and phages are listed in Table 1. The media used for these studies have been previously described (31). Minimal medium was M9 or M63 supplemented with 0.2% glucose (or another carbon source), 1 ,ug of thiamine per ml, and any required L-amino acids (Sigma Chemical Co.) at 40 ,ug/ml. Solid media contained 1.5% agar (Difco Laboratories) or 0.7% agar for soft agar overlays. Lactose indicator plates contained 40 ,ug of X-Gal (5-bromo-4-chloro-3-indolyl-,-D-galactopyranoside; Boehringer Mannheim) per ml in minimal medium containing...
This study evaluated the in vitro activity of ceftaroline, a novel cephalosporin with broad-spectrum activity against gram-negative and -positive pathogens, against 4,151 recent clinical isolates collected in the United States. Ceftaroline was very potent against bacteria found in community-and hospital-acquired infections, including methicillin-resistant Staphylococcus aureus, multidrug-resistant Streptococcus pneumoniae, and common Enterobacteriaceae spp.
Increased ceftaroline MICs were associated with decreased PBP2a binding affinity and reflected alterations in PBP2a.
Ceftaroline fosamil is a novel cephalosporin with broad-spectrum activity against Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Streptococcus pneumoniae, and common Gram-negative organisms. The activity of ceftaroline against MRSA is attributed to its ability to bind to penicillin-binding protein (PBP) 2a with high affinity and inhibit the biochemical activity of PBP 2a more efficiently than other presently available β-lactams. The activity of ceftaroline against MRSA and the β-haemolytic streptococci makes it an attractive monotherapy agent for the treatment of complicated skin and skin structure infections (cSSSIs). Recent profiling and surveillance studies have shown that ceftaroline is active against contemporary skin pathogens collected from US and European medical centres in 2008. The mean free drug %T > MIC (percentage of time the drug concentration remains above the MIC) needed for stasis ranged from 26% for S. aureus to 39% for S. pneumoniae in the murine thigh infection model. Pharmacokinetic and pharmacodynamic target attainment predictions for 600 mg of ceftaroline fosamil every 12 h showed that the mean %T > MICs for which plasma free-drug concentrations exceeded an MIC of 1 and 2 mg/L were 71% and 51% of the dosing interval, respectively. For a 40% T > MIC target, the predicted attainments for infections due to pathogens for which ceftaroline MICs were 1 or 2 mg/L were 100% and 90%, respectively. Clinical and microbiological successes of ceftaroline fosamil in treating cSSSIs were demonstrated in two Phase III clinical studies, in which 96.8% of all baseline cSSSI isolates from the microbiologically evaluable population were inhibited by ceftaroline at ≤ 2 mg/L. Ceftaroline fosamil is a promising broad-spectrum agent for the treatment of cSSSIs.
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